Photodynamic therapy using Photofrin and excimer dye laser treatment for superficial oral squamous cell carcinomas with long-term follow up

ObjectivesPhotodynamic therapy (PDT) is a very effective treatment for superficial malignancies that does not result in loss of normal tissue. Here, we report successful PDT treatment of superficial oral cancers and its clinical outcome with long-term follow up.Materials and methodsThirty-four superficial oral squamous cell carcinomas were treated with PDT, and the effects were evaluated. Each patient received Photofrin (2 mg/kg) intravenously 48 h prior to light irradiation. Photoradiation was performed at doses of 100–150 J/cm² using a 630-nm wavelength excimer dye laser.ResultsSix months after PDT, 30 patients (88.2%) showed complete responses while 9 patients (26.5%) had local relapses during long-term follow-up. The 5-year overall survival, disease-specific survival, and disease-free survival rates were 76.5%, 84.6%, and 63.3%, respectively. Lesions with red patches had a significantly higher recurrence rate than lesions with white patches. Accurate evaluation of the extent of lesions and appropriate photoradiation were important in improving outcomes. Adverse events observed included sunburn and sequestrum formation of alveolar bone. No abnormal laboratory values or systemic complications were observed.ConclusionPDT using Photofrin as the photosensitizer is an effective treatment modality for superficial oral carcinomas, with excellent healing and minimal side effects.     

Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms

Background dataMethylene blue (MB) and toluidine blue (TB) are recognized as safe photosensitizers (Ps) for use in humans. The clinical effectiveness of the antimicrobial photodynamic therapy with MB and TB needs to be optimized, and ethanol can increase their antimicrobial effect. Formulations of MB and TB containing ethanol were evaluated for their ability to produce singlet oxygen and their antibacterial effect on Pseudomonas aeruginosa biofilms.MethodsPhotoactivated formulations were prepared by diluting the Ps (250 μM) in buffered water (pH 5.6, sodium acetate/acetic acid), 10% ethanol (buffer: ethanol, 90:10), or 20% ethanol (buffer: ethanol, 80:20). Biofilms also were exposed to the buffer, 10% ethanol, or 20% ethanol without photoactivation. Untreated biofilm was considered the control group. The production of singlet oxygen in the formulations was measured based on the photo-oxidation of 1,3-diphenylisobenzofuran. The photo-oxidation and CFU (log₁₀) data were evaluated by two-way ANOVA and post-hoc Tukey’s tests.ResultsIn all the formulations, compared to TB, MB showed higher production of singlet oxygen. In the absence of photoactivation, neither the buffer nor the 10% ethanol solution showed any antimicrobial effect, while the 20% ethanol solution significantly reduced bacterial viability (P = 0.009). With photoactivation, only the formulations containing MB and both 10% and 20% ethanol solutions significantly reduced the viability of P. aeruginosa biofilms when compared with the control.ConclusionsMB formulations containing ethanol enhanced the antimicrobial effect of the photodynamic therapy against P. aeruginosa biofilms in vitro.     

Photodecomposition,photomutagenicity and photocytotoxicity of retinyl palmitate under He–Ne laser photoirradiation and its effects on photodynamic therapy of cancer cells in vitro

ObjectiveOur aim was to study photodecomposition, photomutagenicity and cytotoxicity of retinyl palmitate (RP), a principal storage form of vitamin A in humans and animals, under He–Ne laser photoirradiation. Moreover, the effect of different concentrations and timing protocol of antioxidants on photodynamic therapy (PDT) is contradictory, so the effect of RP (as antioxidant) on the PDT cytotoxicity was studied.MethodsPhotomutagenicity was tested by Ames test. Photodecomposition was studied by UV–vis spectroscopy. Cytotoxicity was measured with MTT-assay. Moreover, the effect of PDT, using hematoporphyrin derivatives (HpD) as photosensitizer under He–Ne laser irradiation (10 J/cm²), was studied on HeLa cells either with or without RP (1–100 μM) which incubated with the cells for short or long incubation period (1 h or 24 h) prior to PDT.ResultsNo photodecomposition of RP alone was obseved whereas there is a little photodecomposition of RP only in presence of HpD under irradiation with He–Ne laser. Moreover, no photomutagenicity was observed in Salmonella typhimurium strains under laser irradiation in presence or absence of HpD. RP alone (1–100 μM) significantly decrease the viability of HeLa cells. Laser irradiation of HeLa cells pre-incubated with RP alone for 24 h showed further significant decrease in viability of the cells. While RP incubations for 1 h before PDT had slight effect on the cells, 24 h incubation before PDT enhanced the cytotoxicity of PDT on HeLa cells.ConclusionsRP can be used 24 h before PDT to enhance its effects. RP is not mutagenic under irradiation with He–Ne laser.     

Evaluation of BPA uptake in clear cell sarcoma (CCS) in vitro and development of an in vivo model of CCS for BNCT studies

Clear cell sarcoma (CCS), a rare malignant tumor with a predilection for young adults, is of poor prognosis. Recently however, boron neutron capture therapy (BNCT) with the use of p-borono‐L‐phenylalanine (BPA) for malignant melanoma has provided good results. CCS also produces melanin; therefore, the uptake of BPA is the key to the application of BNCT to CCS. We describe, for the first time, the high accumulation of boron in CCS and the CCS tumor-bearing animal model generated for BNCT studies.     

In vitro assessment of anti-tumorigenic mechanisms and efficacy of NanoALA,a nanoformulation of aminolevulic acid designed for photodynamic therapy of cancer

BackgroundThe development of nanocarriers is an important approach to increase the bioavailability of hydrophilic drugs in target cells. In this work, we evaluated the anti-tumorigenic mechanisms and efficacy of NanoALA, a novel nanoformulation of aminolevulic acid (ALA) based on poly(lactide-co-glycolide) (PLGA) nanocapsules designed for anticancer photodynamic therapy (PDT).MethodsFor this purpose, physicochemical characterization, prodrug incorporation kinetics, biocompatibility and photocytotoxicity tests, analysis of the cell death type and mitochondrial function, measurement of the intracellular reactive oxygen species production and DNA fragmentation were performed in murine mammary carcinoma (4T1) cells.ResultsNanoALA formulation, stable over a period of 90 days following synthesis, presented hydrodynamic diameter of 220 ± 8.7 nm, zeta potential of −30.6 mV and low value of polydispersity index (0.28). The biological assays indicated that the nanostructured product promotes greater ALA uptake by 4T1 cells and consequently more cytotoxicity in the PDT process. For the first time in the scientific literature, there is a therapeutic efficacy report of approximately 80%, after only 1 h of incubation with 100 μg mL⁻¹ prodrug (0.6 mM ALA equivalent). The mitochondria are probably the initial target of treatment, culminating in energy metabolism disorders and cell death by apoptosis.ConclusionsNanoALA emerges as a promising strategy for anticancer PDT. Besides being effective against a highly aggressive tumor cell line, the treatment may be economically advantageous because it allows a reduction in the dose and frequency of application compared to free ALA.     

电离辐射对肺癌A549细胞miR-21体内外表达的影响及意义

目的 研究miR-21在肺癌组织及血清中的表达与肺癌预后及放疗因素的关系,进而探讨电离辐射对人肺癌A549细胞体内外表达miR-21的影响。方法 收集肺癌患者病理组织及血清样本,检测不同病理类型肺癌组织及血清中的miR-21的表达水平,同时检测是否接受放疗的非小细胞肺癌患者血清中miR-21的表达水平,并进行生存分析;以2、4 Gy X射线照射体外培养的A549细胞,并以A549细胞制备裸鼠肺转移癌模型,分别检测A549细胞与裸鼠血清及肺中miR-21的表达水平。结果 肺癌组织中miR-21高表达占60.0%;肺癌血清中miR-21高表达占50.5%,腺癌与鳞癌检出率差异有统计学意义（χ²=5.766,P<0.05）;87例非小细胞肺癌中放疗患者miR-21检出率66.7%显著高于非放疗患者39.6%（χ²=6.321,P<0.05）;Kaplan-Meier法生存分析显示,miR-21高表达患者预后明显低于低表达患者,差异有统计学意义（P<0.05）;Cox回归模型分析显示,miR-21高表达、区域淋巴结转移及放疗均为影响患者预后的独立危险因素。2、4 Gy X射线照射A549细胞后不同时间点miR-21表达显著升高（t=-7.552~-1.206,P<0.05）,miR-21在裸鼠血清及肺组织中的表达水平显著升高（t=-47.845~-2.356,P<0.05）。结论 电离辐射可上调A549细胞体内外miR-21的表达水平,可能与增强A549细胞侵袭转移能力相关。     

Investigation of cell death mechanisms in human lymphatic endothelial cells undergoing photodynamic therapy

BackgroundPhotodynamic therapy (PDT) has been shown to induce ablation and functional occlusion of tumor-associated lymphatic vessels. However, direct effects of PDT on lymphatic endothelial cells (LECs) have not been studied so far. The aim of this study was to elucidate molecular mechanisms of cell death induced by PDT in human LECs.MethodsVerteporfin was used as a photosensitizer to investigate PDT-mediated damage of lymphatic vessels in mice using immunofluorescent staining and stereomicroscopy. In vitro dose-response studies were carried-out with crystal violet staining. Immunofluorescence, flow cytometry, immunoblotting and DNA electrophoresis were used to investigate the mechanisms of cell death in human LECs undergoing PDT.ResultsPDT induced an increase in the number of propidium iodide positive lymphatic endothelial cells in the mouse dermis. In in vitro studies dose-dependent cytotoxic effects of PDT towards LECs were observed. Typical hallmarks of apoptotic cell death, including Annexin V binding, loss of mitochondrial membrane potential, caspase activation, cleavage of PARP as well as DNA fragmentation were observed in LECs when PDT was used at high irradiation conditions, causing >80% cell death. At lower light fluencies causing <50% cell death PDT induced autophagy rather than apoptosis, as revealed by conversion of LC3-I to the autophagosomal LC3-II and formation of LC3 puncta. Z-VAD-FMK, a caspase inhibitor, prevented cell death induced by high-dose PDT only, while 3-methyladenine, an autophagy suppressor, inhibited cell death induced by low-dose PDT.ConclusionsBoth apoptosis and autophagy are involved in cell death induced by verteporfin-PDT in LECs.     

miR-424*在X射线照射后的A549细胞体内、外及非小细胞肺癌组织和血清中的表达

目的 研究2、4 Gy X射线照射后人肺腺癌细胞miR-424^*体内、外表达以及非小细胞肺癌患者肺组织及血清中miR-424^*的表达变化及其意义。方法 2、4 Gy X射线分别照射体外培养的A549细胞,以实时定量PCR（RT-qPCR）法检测A549细胞中miR-424^*表达水平;用照射后的A549细胞制备裸鼠肺转移动物模型,检测裸鼠肺组织及血清中miR-424^*的表达水平;收集肺癌患者肺组织及血清样本,检测miR-424^*表达水平。结果 2、4 Gy X射线照射后1、2、12、24及48 h,miR-424^*表达均显著升高（2 Gy：t=-45.886~-6.709,P<0.05;4 Gy：t=-29.087~-7.833,P<0.05）;0、2、4 Gy照射后,miR-424^*在裸鼠肺及血清中表达水平分别为空白对照组的9.72、8.58及4.7与11.93、9.22及8.99倍（t=-13.243~-3.052,P<0.05）。6/11例（54.5%）患者肺癌组织中高表达miR-424^*,腺癌、鳞癌病理类型间检出率差异无统计学意义（P>0.05）;43/84例（51.20%）肺癌患者与健康志愿者相比,血清miR-424^*表达升高 1.97~17.71倍,其中腺癌患者血清检出率为39.1%（18/46）,鳞癌患者血清检出率为65.8%（25/38）,两种病理类型检出率差异有统计学意义（t=5.919,P<0.05）;此外,84例肺癌患者中,miR-424^*[JP3]在未接受放疗的肺癌患者血清中的阳性检出率为41.5%（22/53）,显著低于接受放疗的肺癌患者血清的阳性检出率67.7%（21/31）[JP]（t=5.387,P<0.05）。结论 2、4 Gy X射线照射可增加A549细胞miRNA-424^*的体内、外表达水平,可能与增强A549细胞体内、外侵袭转移能力有关。肺癌患者中50%以上的肺癌组织及肺癌患者血清中miR-424^*表达水平显著升高,可能与肺癌的病理类型及放疗相关。     

变态反应性接触性皮炎体内外模型制作探讨

 目的 建立变态反应性接触性皮炎(allergic contact dermatitis,ACD)体内外模型。方法 采用二硝基氟苯(2,4-dinitrofluorobenzene,DNFB)建立小鼠耳廓ACD模型,计算耳肿胀度、HE染色;离体培养ACD小鼠T淋巴细胞,MTT法分析细胞增殖活性,免疫荧光检测CD4的表达水平,双抗体夹心ELISA法检测细胞因子IL-2和IL-4的含量。结果 小鼠ACD模型耳可见明显肿胀、大量炎性反应细胞浸润;模型小鼠T淋巴细胞体外培养能维持一定时程的高反应性及CD4的高水平表达,IL-2的峰值显著高于IL-4峰值。结论 成功建立了简单、快速、可靠的体内外ACD模型,可为评估变应原对人类健康的风险及筛选抗ACD免疫调节新药积累实验依据。     

光动力疗法辅助手术治疗脑肿瘤的随访研究

本文报告应用PDT辅助手术治疗脑肿瘤30例,其中9例为复发性胶质瘤。用HpD4～7mg/kg体重,和激光波长630nm,剂量达到300J/cm~2辅助手术治疗,取得满意效果,且未增加并发症。经随访,平均生存时间已达18.5个月;1、2和3年的生存率分别达到50％、29％和25％。对照组平均生存时间仅达12.5个月;1、2和3年的生存率均为21％。证明PDT辅助手术治疗脑肿瘤优于单纯手术治疗。     

Photodynamic therapy activated STAT3 associated pathways: Targeting intrinsic apoptotic pathways to increase PDT efficacy in human squamous carcinoma cells

5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) has been used for part of squamous cell carcinoma (premalignant conditions or in situ cutaneous SCC–Bowen disease). However, mechanism of ALA-PDT is not fully understood yet on the cell apoptosis pathway. The aim of this study was to further investigate the effect and mechanism of 5-ALA-PDT on human squamous carcinoma A431cells. Apoptosis and cell viability after PDT were evaluated using Annexin V-FITC apoptosis detection kit and MTT assay. The mRNA and protein levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Our data showed that 5-ALA-PDT significantly inhibited cell proliferation (p< 0.05), but there was no significant difference when the photosensitizer reached to 4.8 mM. The inhibition in cell proliferation after 5-ALA-PDT treatment was correlated to more cells being arrested in the G0/G1 phase of the cell cycle (p < 0.01). Immunocytochemical observations using anti-active caspase-3 antibodies showed active caspase-3 was translocated from cytoplasm to nuclear during apoptosis. STAT3 and its downstream gene Bax and BCL-2 were changed after 5-ALA-PDT treatment for the mRNA and protein expression. Our studies confirmed that 5-ALA-PDT might be an effective treatment for human squamous carcinoma by inhibiting the tumor cell A431growth and for the first time demonstrated that the expression of STAT3 was significantly reduced at 24 h after 5-ALA-PDT treatment.     

光动力疗法与热损伤对正常大鼠胃损伤的研究

为了初步探讨ＰＤＴ与热损伤对正常胃损伤作用机制的差异，本实验以血卟啉衍生物及氩离子泵染料激光器为光敏剂及光源，观察了大鼠胃壁在单纯氩离子激光照射和ＰＤＴ时，照射部位的温度及组织学在照射后７２小时和二周的改变。结果：氩离子激光照射组和ＰＤＴ组胃壁温度均高于对照组（Ｐ＜０．０１），但ＰＤＴ组温度均低于３７℃。氩离子激光照射后的胃壁组织在７２小时各层组织均明显出血，炎细胞浸润，组织凝固坏死。电镜下见胶原纤维肿胀，排列紊乱，甚至溶解吸收。二周后损伤部位以纤维瘢痕组织修复。ＰＤＴ组在７２小时胃壁组织也有充血和炎细胞浸润，但仅有轻度组织坏死，损伤深度未超过粘膜下层。电镜下见胶原纤维结构基本正常。二周后损伤部位以组织再生修复，未留瘢痕。结论：ＰＤＴ与氩离子激光组织损伤及修复方式不同，ＰＤＴ不损伤胃粘膜下胶原纤维。     

激光功率密度对HMME-PDT的视网膜和脉络膜生物学效应的影响

目的观察激光功率密度对血卟啉单甲醚(HMME)的视网膜和脉络膜的光动力效应的影响。方法以新西兰兔正常脉络膜毛细血管层为实验对象,HMME注射剂量为5mg/kg,波长532nm激光作为激发光源,眼底光斑能量密度为20J/cm2,功率密度分别为100、200、300和400mW/cm2,相匹配的照射时间分别为200、100、66和50s,照光时机为注射药物结束后5min之内。在PDT后第6小时和12小时,1、3和5天进行眼底观察、荧光眼底造影,并在眼底造影出现脉络膜毛细血管闭塞时取照光部位进行组织学检查。结果激光功率密度为100mW/cm2,PDT后第5天出现脉络膜毛细血管的选择性闭塞;功率密度为200mW/cm2时,PDT后第3天出现脉络膜毛细血管的选择性闭塞;功率密度为300mW/cm2时,PDT后第1天出现视网膜的非选择性损伤,PDT后第3天视网膜恢复正常,脉络膜毛细血管闭塞;功率密度为400mW/cm2时,PDT后第6h出现视网膜的非选择性损伤,PDT后第5天视网膜恢复正常,脉络膜毛细血管和大部分脉络膜大血管的闭塞。结论在激光能量密度相同时,其功率密度是影响生物学效应的重要因素。即随着功率密度的增加,生物学效应出现的时间提前,照射部位的视网膜和脉络膜大血管受累的可能性越大。